In order to maintain its protective function, the epidermis undergoes a continual process of homeostatic renewal in which cells within the proliferative basal layer withdraw from the cell cycle and differentiate as they process through the suprabasal epidermal layers. The morphological and genetic changes associated with epidermal keratinocyte differentiation are well described. How other molecular mechanisms, such as metabolic reprogramming regulate epidermal homeostasis, have not been extensively studied. We recently demonstrated that oxidative mitochondrial metabolism and reactive oxygen species (ROS) production are critical regulators which promote keratinocyte differentiation. Mice which lacked mitochondrial metabolism and ROS generation in basal epidermal cells exhibited impaired epidermal differentiation and increased levels of proliferative basal cells. We further demonstrated that ROS produced at mitochondria act as critical signaling molecules which promote the propagation of Notch signaling events which are required for keratinocyte differentiation. In the current proposal, we will build on our recently-reported findings to dramatically extend our understanding of how the epidermal differentiation program regulates cellular metabolism to promote differentiation or to maintain basal cells in the undifferentiated state. We will focus our attention on p63, a transcription factor required for epidermal development and homeostasis. p63 belongs to a family of transcription factors which are known to be regulators of cellular metabolism and ROS levels. We will use gain and loss of function studies to determine how p63 expression affects keratinocyte glycolytic and oxidative metabolism. We will closely examine how p63 expression affects metabolite flux through biosynthetic pathways and how p63 expression affects levels of cellular ROS. We will also explore the known repressive effect that p63 has on Notch. We will determine if p63 regulates Notch through repression of ROS-mediated signaling. Our studies will determine how keratinocyte metabolism changes during differentiation and will determine the causal role that metabolic reprogramming plays in promoting differentiation. The work proposed herein will provide the applicant with the training required for development into an independent skin biologist. This proposal builds off of the existing strengths of the applicant and adds to his abilities with additional training in epidermal and epithelial biology. The collaborative environment present at Northwestern University will ensure that the work is completed expeditiously and efficiently. Importantly, the training provided for in this proposal will allow applicant time to take advantage of opportunities for career development available at Northwestern University. These include workshops in grant writing, mentoring, teaching, and responsible conduct of research.